This proposal is in response to RFA-RM-06-010, Using Metabolomics to Investigate Biological Pathways and Networks. We quote from the RFA: "The goal of the current funding opportunity is to apply metabolomics technology to the study of pathway and network regulation in normal and disease states that are not easily approached by other technologies." This proposal employs metabolic cytometry to monitor glycolipid metabolism in single neurons. In metabolic cytometry, fluorescently labeled substrate is incubated with cells and is metabolized to a suite of catabolic and anabolic products. These products are analyzed in a single cell by capillary electrophoresis with ultrasensitive laser-induced fluorescence. As long at the fluorescent tag remains intact, all metabolic products can be detected at the zeptomole to yoctomole level. Errors in glycolipid metabolism are important in a number of serious neurodegenerative diseases, and there is ample evidence that there is dramatic cell-to-cell variation in glycolipid expression. This proposal will use highly innovative tools to characterize glycolipid metabolism in single cells. The project requires a significant synthetic effort to prepare fluorescent substrates and reagents. The project requires additional analytical technology development to simultaneously monitor glycolipid metabolism along different paths. The project requires significant biological effort to use these technologies to monitor glycolipid metabolism across populations of cultured cells and primary neurons. Ole Hindsgaul and Monica Palcic, at the Carlsberg Laboratory, serve as synthetic chemist and biochemist. They will prepare a suite of fluorescently-labeled gangliosides and members of the globoseries of glycolipids. They will also verify that these reagents are substrates for endogenous enzymes in the biosynthetic and biodegradation pathways. Norman Dovichi of the University of Washington will develop ultrasensitive capillary electrophoresis/laser-induced fluorescence technology to monitor these compounds in single cells at the low zeptomole level. Ronald Schnaar (Johns Hopkins) will incubate fluorescent substrates with primary nerve cells and embryonic stem cells at different stages of nerve cell determination, differentiation and maturation. The treated cells will be fixed and transferred to Dovichi's lab for analysis.